Active microneedles and microneedle arrays

ABSTRACT

An active needle device for fluid injection or extraction includes at least one hollow elongated shaft defining at least one channel. The channel provides communication between at least one input port and at least one output port of the needle device. At least one active component such as a sensor or actuator is placed or integrated into the elongated shaft. The needle device can include a macroneedle, a microneedle, or an array of macroneedles or microneedles. The microneedles can be fabricated on a substrate which can remain attached to the microneedles or be subsequently removed. The active component can facilitate biochemical, optical, electrical, or physical measurements of a fluid injected or extracted by the needle device.

RELATED APPLICATIONS

This application claims priority to, the benefit of, and is a divisionalof, U.S. patent application Ser. No. 10/258,011, entitled “IntegratedNeedle Devices with Integrated Functionality,” filed Feb. 23, 2004,which application is expressly incorporated herein by this reference, inits entirety. This application is also related to U.S. Pat. No.7,048,723, entitled “Surface Micromachined Microneedles,” issued on May23, 2006, and U.S. Provisional Application No. 60/101,064, filed on Sep.18, 1998, each of which are expressly incorporated herein by thisreference, in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to needle devices for theinjection and extraction of fluids. More specifically, the presentinvention relates to active needle devices such as macroneedles,microneedles, and macroneedle or microneedle arrays which have one ormore active components providing additional functionality to the needledevices.

2. Relevant Technology

Micro instrumentation is a rapidly growing area of interest for a broadspectrum of applications. One particularly fast growing area isbiomedical instrumentation where significant efforts are being made todevelop micro biochemical analysis systems, physiological analysissystems, and drug delivery systems. A variety of manufacturingtechnologies are used to fabricate these micro systems, many of whichare categorized under the set of technologies known as micromachining.The number of biomedical applications for micromachining technologies israpidly growing. Since micromachining technologies are relatively new,there is an increasing set of manufacturing techniques and criticalapplications still to be addressed.

It is well known that needles are used to extract samples of substancesfrom living tissue in order to analyze the substances for diagnosticpurposes, or to deliver a drug or medicament. The majority of needles inuse today are macroneedles, which have a relatively large diameter ascompared to a blood cell and are on the order of millimeters (mm).

In many areas of biotechnology and medicine, there exists the need forfluid injection or extraction on a microscale; either for injection intoa precise location, or for injecting or extracting small amounts offluid. It is advantageous to be able to perform these injections orextractions with a minimal amount of tissue damage, and also with aminimum amount of discomfort and pain to patients. Microneedles andmicroneedle arrays are capable of performing these tasks. For example,microneedles and microneedle arrays can be used as preciseinjection/extraction needles in cell biology, as injection/extractionheads in a drug delivery system or microchemical factory, and asinjection/extraction heads in microsurgery. Some of the smallest hollowneedles that are currently available have inner diameters of over 200μm. Prior micro-sized (sizes on the order of microns, where 1 micron=1μm=10⁻⁶ m) needles include those disclosed in U.S. Pat. No. 5,457,041 toGinaven et al., and U.S. Pat. No. 5,591,139 to Lin et al.

For some applications, it is desirable to inject small amounts of fluid;however, in other situations, larger amounts of fluid are required to beinjected. Most of the prior systems do not have the capability totransmit larger amounts of fluid into a precise location. One of themethods used to address this problem is to fabricate an array ofneedles, as in U.S. Pat. No. 5,457,041 referred to hereinabove, whichdiscloses an array of microneedles of about 20 needles by 20 needles,wherein the length of the needles is between 10 and 25 microns, and thespacing between needles is between about 5 and 20 microns.

In U.S. Pat. No. 5,591,139 referred to above, silicon-based microneedlesare disclosed which are fabricated using integrated circuit processes.Various devices such as microheaters, mircrodetectors, and other devicescan be fabricated on the microneedle.

Problems with prior microneedles include relatively poor mechanicaldurability. This is mainly due to the fact that such microneedles havebeen made out of etched silicon or out of chemical vapor depositedpolysilicon, both of which have a tendency to be brittle and breakeasily.

In some cases, it is desirable to analyze the fluids being injected orextracted by the microneedle(s). Prior microneedles generally have beenseparate from the systems used to analyze (chemically, optically, orotherwise) the fluids. In these cases, having a separate analysis systemcan require additional equipment and is costly, complex, andinconvenient.

It would therefore be of substantial interest to develop a durableneedle device which is capable of injecting or extracting precisequantities of fluids into specific locations with a minimal amount oftissue damage, and which has integrated sensing capabilities.

SUMMARY OF THE INVENTION

Active needle devices of the present invention have integratedfunctionalities such as biochemical, electrical, or optical sensingcapabilities. The active needle devices can be active macroneedles ormicroneedles, as well as active macroneedle or microneedle arrays, whichincorporate biosensors therein such as for monitoring metabolic levels.

In one embodiment of the invention, an active needle device for fluidinjection or extraction includes at least one hollow elongated shaftdefining at least one channel therethrough. The channel providescommunication between at least one input port and at least one outputport of the needle device. At least one active component such as asensor or actuator is placed or integrated into the elongated shaft. Theneedle device can be a macroneedle, a microneedle, or an array ofmacroneedles or microneedles. The microneedles can be fabricated on asubstrate which can remain attached to the microneedles or can besubsequently removed. The active component can facilitate biochemical,optical, electrical, or physical measurements of a fluid injected orextracted by the needle device.

Two- or three-dimensional microneedle arrays can be constructed havingcross-coupling flow channels that allow for pressure equalization, andbalance of fluid flow within the microneedle arrays. A plurality ofmechanical support members can be integrated into the arrays forstability and to control the penetration depth of the microneedles. Inaddition, an active microneedle or microneedle array may include variousfunctionalities such as integrated biochemical sensors, as well aselectrical, optical, or mechanical transducers and sensors. The sensorsreduce or eliminate the requirement for additional external analysisequipment and enhance overall device portability, disposability, andcompactness.

The active microneedle and microneedle arrays of the invention areparticularly useful for fluid extraction and analysis. In oneembodiment, an active microneedle or microneedle array includesbiochemical sensing reagents which are deposited on an inner surface ofthe microneedle(s), with a window provided on a surface of themicroneedle(s) for the detection of bioluminescence which occurs in thepresence of certain metabolic substances.

A method of fabricating an active microneedle device according to thepresent invention includes providing a substrate with a substantiallyplanar surface and depositing a metal material on the planar surface toform one or more bottom walls for one or more microneedles. A topsurface of the bottom walls is coated with a photoresist layer to aheight corresponding to a selected inner height of a microchannel foreach of the microneedles. A metal material is then deposited to formside walls and a top wall upon the bottom walls and around thephotoresist layer. The photoresist layer is then removed from eachmicrochannel to form the microneedles. The microneedles can be releasedfrom the substrate and used independently of the substrate, if desired.

The method of fabricating the active microneedle device can include p⁺etch-stop membrane technology, anisotropic etching of silicon inpotassium hydroxide, sacrificial thick photoresist micromoldingtechnology, and micro-electrodeposition technology. For a biochemicalluminescence sensing microneedle device, certain biochemical sensingreagents can be drawn into and dried onto the inner walls of the hollowneedle(s).

The active needle devices of the invention have the advantage ofpermitting real-time analysis of a fluid being sampled. In addition, thearray of active microneedles are capable of injecting or extractingrelatively large quantities of fluids with minimal tissue damage and canbe readily attached to a standard syringe. The array of activemicroneedles or a single active microneedle can also be easily andeconomically fabricated. Yet another advantage of the array of activemicroneedles or a single active microneedle is a high degree ofmechanical durability.

These and other features of the present invention will become more fullyapparent from the following description, or may be learned by thepractice of the invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more fully understand the manner in which the above recitedand other advantages of the invention are obtained, a more particular,description of the invention briefly described above will be rendered byreference to specific embodiments thereof which are illustrated in theappended drawings. Understanding that these drawings depict only typicalembodiments of the invention and are not therefore to be consideredlimiting of its scope, the invention will be described and explainedwith additional specificity and detail through the use of theaccompanying drawings in which:

FIG. 1A is a schematic representation of a single active needle deviceaccording to the present invention;

FIG. 1B is a schematic representation of a portion of a single activeneedle device in the form of a microneedle according to one embodimentof the present invention;

FIGS. 2A and 2B are schematic depictions of multi-lumen activemicroneedles according to alternative embodiments of the presentinvention;

FIG. 3 is a schematic representation of microneedles in atwo-dimensional array according to a further embodiment of the presentinvention;

FIG. 4 is a schematic representation of microneedles in atwo-dimensional array according to another embodiment of the presentinvention;

FIG. 5 is a schematic representation of microneedles in atwo-dimensional array according to another embodiment of the presentinvention;

FIGS. 6A-6B are schematic depictions of single-lumen active microneedlearrays according to alternative embodiments of the present invention;

FIGS. 7A-7B are schematic depictions of multi-lumen active microneedlearrays according to alternative embodiments of the present invention;

FIGS. 8A-8F schematically depict the fabrication process sequence forforming a microneedle array,

FIG. 8G depicts an embodiment of a multi-lumen microneedle formedaccording to the present invention;

FIGS. 9A and 9B depict alternative methods of assembling two-dimensionalneedle arrays into three-dimensional needle array devices;

FIG. 10 is a schematic depiction of a pressure sensor in an activemicroneedle according to the invention;

FIG. 11 is a graph of the normalized bioluminescent signal as a functionof glucose concentration;

FIG. 12 is a graph of the normalized bioluminescent signal as a functionof time for several different concentrations of creatine;

FIG. 13 is a graph of the normalized bioluminescent signal as a functionof creatine concentration; and

FIG. 14 is a graph of the average CCD counts as a function of volume fordifferent sample volumes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to active needle devices, such asmacroneedles, microneedles, and macroneedle or microneedle arrays, whichhave one or more active components incorporated therein providingadditional functionality to the needle devices. An active needle deviceaccording to the invention generally includes at least one hollowelongated shaft defining at least one channel therethrough The channelprovides communication between at least one input port and at least oneoutput port of the needle device. At least one active component such asa sensor or actuator is placed or integrated into the elongated shaft.The active component can facilitate biochemical, optical, electrical, orphysical measurements of a fluid injected or extracted by the needledevice. The active component also reduces or eliminates the requirementfor additional external analysis equipment, and enhances the overalldevice portability, disposability, and compactness.

Referring to the drawings, wherein like structures are provided withlike reference designations, FIG. 1A is a schematic representation of asingle active needle device 10 according to one embodiment of thepresent invention. The needle device 10 includes a hollow elongatedshaft 11 defining a single channel 12 from a proximal inlet end 13 to adistal tip end 14 which is tapered. One or more input ports 15 arelocated at the proximal inlet end 13, of elongated shaft 11 and one ormore output ports 16 are located toward the distal tip end 14. Thechannel 12 provides communication between input ports 15 and outputports 16.

The needle device 10 can be either a macroneedle or a microneedle,depending on the configuration of elongated shaft 11 defining channel12. A “macroneedle” as used herein means any conventional type needleused to inject or extract fluids, such as conventional metallic needlesused in medical applications. A “microneedle” as used herein means anymicro-sized needle which can be generally fabricated by micromachiningtechniques.

At least one active component 17 is placed or integrated directly inelongated shaft 11. The active component 17 can include one or moreelectronic components, electromechanical components, mechanicalcomponents, biochemical components, or various combinations thereof.These components can be configured to form one or more sensors,actuators, or combinations thereof. For example, the sensors can be abiosensor, such as a bioluminescence-based biosensor, which is discussedin further detail hereafter, microdetectors, optical pressure sensors,DNA analyzer chips, and the like. The actuators can includeelectromechanical components for fluid manipulation such as micropumps,microvalves, or the like. In addition, active component 17 can includean integrated circuit chip, an optical detector, other electrical basedcomponents, impedance analyzers (e.g., integrated resistance,inductance, and capacitance sensors), transducers, and the like. Atransducer can be used for detection of fluid parameters such as flowvelocity, temperature, pressure, etc. The active component 17 canfacilitate one or more biochemical, optical, electrical, or physical,measurements or other analyzing functions for a fluid injected orextracted by needle device 10.

The active component 17 can be interfaced electronically withconventional analyzing equipment or can be a stand alone device. Inaddition, the active component 17 may be fabricated independently of theneedle device and subsequently placed therein, or may in some cases befabricated at the same time as the needle device. If fabricatedindependently, the active component can be made on separate chips, whichcan be affixed to an internal surface of the needle or substituted forpart of a wall of the needle. Additional details related to activecomponent 17, including specific embodiments thereof, are discussedbelow.

The active component 17 allows the needle device to provide real-timeanalysis of a fluid being sampled such as in a blood analysis system.For example, blood can be analyzed by an on-chip amplifier, such as ablood analysis amplifier, and an A/D converter which converts an analogsignal to a digital signal for transmission to a computer for real-timecomputer analysis.

When needle device 10 is configured as a single microneedle, elongatedshaft 11 generally includes a bottom wall formed by a solid layer 19,and opposing side walls connected by a top wall which are formed by ahollow layer 20, as shown in FIG. 1B. These bottom, side, and top wallsdefine channel 12 in the form of a microchannel with a transversecross-sectional profile that is substantially rectangular. Themicroneedle optionally has a flange 18 at proximal inlet end 13 (FIG.1A) which functions as a structural support for the microneedle. Theflange 18 can control penetration depth and can be used to mechanicallyfix the microneedle to a surface that is penetrated. The input ports 15located at proximal inlet end 13 and output ports 16 are formed in oneor more of the bottom wall, side walls, or top wall of the microneedle.The input ports 15 and output ports 16 in the microneedle can be formedby conventional fabrication processes such as etching. FIG. 1B showsactive component 17 such as a biosensor adjacent to output ports 16.

The microneedle can be fabricated by micromachining techniques from avariety of metallic materials such as nickel, copper, gold, silver,platinum, palladium, titanium, chromium, alloys thereof, and the like,as well as other materials such as silicon, polysilicon, ceramics,glass, carbon black, plastics, composites thereof, etc. In oneembodiment, the microneedle is fabricated from a non-silicon materialsuch as any of the above metallic materials using the micromachiningtechniques discussed in further detail below. The microneedle can befixed on a substrate where fabricated or can be removed from a substrateafter fabrication.

FIGS. 2A and 2B are schematic depictions of multi-lumen activemicroneedles according to alternative embodiments of the presentinvention. FIG. 2A illustrates a dual-lumen active microneedle 21 whichis constructed in a similar manner as the microneedle of FIG. 1B, havingan elongated shaft 11 including a bottom wall formed by a solid layer19, and opposing side walls connected by a top wall which are formed bya hollow layer 20. The hollow layer 20 is configured such thatmicroneedle 21 has two microchannel members 22 a and 22 b each with alumen therein and each with corresponding inputs 23 a and 23 b. One ormore active components 17 such as biosensors are located in each ofmicrochannel members 22 a and 22 b.

FIG. 2B illustrates a tri-lumen active microneedle 24 which isconstructed in a similar manner as microneedle 21. The microneedle 24has an elongated shaft 11 including a bottom wall formed by a solidlayer 19, and opposing, side walls connected by a top wall which areformed by a hollow layer 20. The hollow layer 20 is configured such thatmicroneedle 24 has three microchannel members 22 a, 22 b, and 22 ctherein, each with corresponding inputs 23 a, 23 b, and 23 c. One ormore active components 17 such as biosensors are located in each ofmicrochannel members 22 a, 22 b, and 22 c.

The multi-lumen active microneedles according to the present inventionallow two or more different fluids to be delivered by the microneedle,which are mixed just before delivery. The multi-lumen activemicroneedles can also be used to separately distribute an extractedfluid for multiple analyses.

FIG. 3 is a schematic depiction of an active microneedle array 25 aaccording to another embodiment of the invention. The microneedle array25 a is formed in a two-dimensional configuration on a substrate 26having a substantially planar upper surface 27. The substrate 26 ispreferably composed of a semiconductor material such as silicon,although other materials can be employed such as glass, metals,ceramics, plastics, and composites or combinations thereof.

A plurality of hollow microneedles 28 are formed on upper surface 27 ofsubstrate 26. The microneedles 28 each have a bottom wall, two opposingside walls, and a top wall that define a microchannel therein. Eachbottom wall is formed partially on upper surface 27 of substrate 26. Themicroneedles 28 each have one or more input ports 15 at a proximal endthereof. A distal portion of each microneedle 28 including themicrochannels extends beyond an edge of upper surface 27 of substrate 26in a cantilevered manner and terminates in needle tips 29 which can havea channel opening therein. In addition, one or more outlet ports 16 canbe located adjacent to needle tips 29. The microneedles 28 arepreferably aligned substantially parallel to each other on substrate 26.One or more active components 17, such as biosensors or other sensing oractuating devices as discussed above, are located in each ofmicroneedles 28.

The microchannels in the microneedles 28 are preferably dimensioned tohave a width between sidewalls of less than about 100.mu.m, and morepreferably about 0 μm to about 50 μm. When the width is zero between thesidewalls, the microneedles 28 effectively become one multi-lumenmicroneedle with a plurality of microchannels as shown in FIGS. 2A and2B. The height between the top and bottom walls of the microchannel isalso preferably less than about 100 μm, and more preferably about 2 μmto about 50 μm. The length of each microneedle can be from about 0.05 μmto about 5 mm, and the width of each microneedle can be from about 0.05μm to about 1 mm. The center-to-center spacing between individualmicroneedles can be from about 50 μm to about 200 μm. The microneedlescan also withstand flow rates of up to about 1.5 cc/sec.

The microneedle length extended from substrate 26 can be varied fromless than about 50 μm (subcutaneous) to several millimeters for fluiddelivery/extraction. For example, tie distal end of each microneedle 28can extend beyond the edge of substrate 26 a distance from about 10 μmto about 100 mm. The inner cross-sectional dimensions of themicrochannels in individual microneedles can range from about 10 μm toabout 1 mm in width and from about 2 μm to about 50 μm in height.Accordingly, the microchannel in each of the microneedles can have across-sectional area in the range from about 25 μm² to about 5000 μm².

A needle coupling channel member 30 can be optionally formed on uppersurface 27 of substrate 26 between microneedles 28. The coupling channelmember 30 has a bottom wall, two opposing side walls, and a top wallthat define a coupling microchannel therein, which provides for fluidcommunication between the microchannels of each microneedle 28. Thecoupling channel member 30 also allows for pressure equalization andbalance of fluid flow between microneedles 28.

Structural support members 31 can be formed on either side of couplingchannel member 30 on upper surface 27 of substrate 26. The structuralsupport members 31 mechanically interconnect microneedles 28 to providerigidity and strength to microneedle array 25 a. The support members 31also precisely control the penetration depth of microneedles 28.

The microneedles 28, coupling channel member 30, and support members 31can be formed from a variety of metal materials such as nickel, copper,gold, palladium, titanium, chromium, alloys or combinations thereof, andthe like, as well as other materials such as silicon, polysilicon,ceramics, glass, carbon black, plastics, composites or combinationsthereof, and the like. The microneedles 28 can be in fluid communicationwith a single fluid input device or with multiple fluid input devices(not shown).

An active microneedle array 25 b according to an alternative embodimentof the invention is shown in FIG. 4. The microneedle array 25 b has atwo-dimensional configuration with similar components as microneedlearray 25 a discussed above, except that the substrate has been removedfrom the array. Accordingly, microneedle array 25 b includes a pluralityof microneedles 32 with microchannels that are dimensioned as discussedabove for microneedle array 25 a.

The microneedles 32 each terminate at a needle tip 33 with a channelopening 34 therein. A needle coupling channel member 36 with a couplingmicrochannel therein provides a fluidic interconnection between themicrochannels of each microneedle 32. A pair of structural supportmembers 38 are formed on either side of coupling channel member 36 andinterconnect with microneedles 32. One or more input ports 37 and outputports 39 can be optionally formed in microneedles 32 to increase fluidinput and output flow. The input ports 37 and output ports 39 can beformed in one or more of the bottom walls, aside walls, of microneedles32 by conventional fabrication processes such as etching. One or moreactive components 17, such as biosensors or other sensing or actuatingdevices as discussed above, are located in each of microneedles 32.

An active microneedle array 40 according to another embodiment of theinvention is shown in FIG. 5. The microneedle array 40 has atwo-dimensional configuration with similar components as microneedlearray 25 b, except that microneedle array 40 is formed without acoupling channel member. Accordingly, microneedle array 40 includes aplurality of microneedles 32 with microchannels that are dimensioned asdiscussed above for microneedle array 25 a. The microneedles 32 eachterminate at a needle tip 33 with a channel opening 34 therein. A pairof structural support members 38 interconnect with microneedles 32. Oneor more input ports 37 and output ports 39 can be optionally formed inmicroneedles 32 to increase fluid input and output flow. One or moreactive components 17, such as biosensors or other sensing or actuatingdevices as discussed above, are located in each of microneedles 32.

FIGS. 6A-6B and 7A-7B are schematic depictions of single and multi-lumenactive microneedle arrays according to alternative embodiments of thepresent invention. An active microneedle array 42 a is shown in FIG. 6Ahaving a two-dimensional configuration with similar components asmicroneedle array 25 b discussed above. Accordingly, microneedle array42 a includes a plurality of microneedles 32 each with single lumenmicrochannels 43. The microneedles 32 each terminate at a needle tip 33.A needle coupling channel member 36 with a coupling microchannel 44therein provides a fluidic interconnection between microchannels 43 ineach microneedle 32. A structural support member 38 interconnectsmicroneedles 32. One or more input ports 37 and output ports 39 arelocated in microneedles 32. One or more active components 17 are locatedin each of microneedles 32.

Another active microneedle array 42 b is shown in FIG. 6B. Themicroneedle array 42 b has a two-dimensional configuration with similarcomponents as microneedle array 42 a, except that microneedle array 42 bis formed without a coupling channel member. Accordingly, microneedlearray 42 b includes a plurality of microneedles 32 each with singlelumen microchannels 43. The microneedles 32 each terminate at a needletip 33 and a structural support member 38 interconnects microneedles 32.One or more input ports 37 and output ports 39 are located inmicroneedles 32, and one or more active components 17 are located ineach of microneedles 32.

FIG. 7A depicts an active microneedle array 45 a having atwo-dimensional configuration with similar components as microneedlearray 42 a discussed above, except that microneedle array 45 a has aduel lumen microchannel configuration. Accordingly, each microneedle 32in array 45 a is constructed to have a pair of microchannels 46 a and 46b. A needle coupling channel member 36 with a coupling microchannel 47therein provides a fluidic interconnection between microchannels 46 aand 46 b in each microneedle 32. One or more input ports 37 and outputports 39 are located in microneedles 32, and one or more activecomponents 17 can be located in each microchannel of microneedles 32.

FIG. 7B depicts an active microneedle array 45 b having atwo-dimensional configuration with similar components as microneedlearray 42 b discussed above, except that microneedle array 45 b has aduel lumen microchannel configuration. Accordingly, each microneedle 32in array 45 b is constructed to have a pair of microchannels 46 a and 46b. A structural support member 38 interconnects microneedles 32, and oneor more input ports 37 and output ports 39 are located in microneedles32. One or more active components 17 can be located in each microchannelof microneedles 32.

In other alternative embodiments, active microneedle arrays can befabricated to have additional microchannels in each microneedle. Forexample, each microneedle in the array can have a tri-lumen channelconstruction such as shown for the microneedle of FIG. 2B discussedabove.

It should be understood that the above discussion related to microneedlearrays is equally applicable to macroneedles, which can be configured invarious arrays. The macroneedle arrays can have similar structures asdescribed above for the microneedle arrays such as single or multi-lumenchannel configurations, multiple input and output ports, structuralsupport members, coupling channel members, and active components in themacroneedles.

A method of fabricating a two-dimensional microneedle array according tothe invention is depicted schematically in FIGS. 8A-8F. As shown in FIG.8A, a substrate 26 having a substantially planar surface 27 is provided,such as a silicon wafer which is polished on both sides. The wafer canhave a thickness of about 1 μm to about 700 μm, and is preferably about150 μm thick. One side of the wafer is heavily doped with boron usinghigh temperature thermal diffusion in order to form a 3-5 μm thick p+layer. Next, silicon nitride (Si₃N₄) is deposited on both sides of thewafer using plasma-enhanced chemical vapor deposition (PECVD). Thesilicon nitride on the undoped side of the wafer is patterned and etchedemploying photoresist as a mask, and then isotropic etching (a CF₄plasma for example) is used to etch the exposed silicon nitride todefine the area upon which the microneedles are to be fabricated. Afterpatterning the silicon nitride layer, the exposed silicon isanisotropically etched using a potassium hydroxide (KOH) solution. Thep+ boron layer serves as an etch stop, resulting in a thin sacrificialmembrane 52, as shown in FIG. 8A. The sacrificial membrane 52 comprisesthe surface upon which the microneedles are formed and then subsequentlyreleased as described below.

Next, a metal system of adhesion layers and an electroplating seed layerare deposited (by electron beam evaporation, for example) onto theinsulating silicon nitride film. The adhesion and seed layers aretypically composed of, but not limited to, titanium, chromium, copper,or combinations thereof. Then, using a mask of the appropriatedimensions and standard photolithographic techniques, this metalmultilayer is patterned. A metal material is then electroplated to formone or more bottom walls 54 (e.g., about 20 μm thick) for themicroneedles, as shown in FIG. 8B. Palladium is one preferred metal forthe bottom wall since it provides high mechanical strength anddurability, is corrosion resistant, provides biocompatibility for use inbiomedical applications, and is easily electroplated to within precisedimensions. Other materials which fit the criteria mentioned could alsobe used equally well, such as copper, nickel, or gold. When performingthe electroplating, the bath chemistry and the electroplating conditions(such as amount of applied current and time in the electroplating bath)should be precisely controlled for optimum results. The typicaldimensions for bottom walls 54 are about 10-20 μm in thickness, andabout 50 μm wide.

After bottom walls 54 have been formed, a commercially available thickphotoresist is deposited (e.g., about 20 μm thick) and patterned usingultraviolet exposure and alkaline developer, resulting in sacrificiallayers 56 as depicted in FIG. 8C. Next, a metal seed layer such as goldis sputter deposited (e.g., about 800 Å thick) onto the photoresistsacrificial layers 56. The metal seed layer serves as an electricalcontact for the subsequent metal electroplating.

A metal layer such as palladium is then electroplated (e.g., about 20 μmthick) onto sacrificial layers 56 to form a plurality of side walls 58and top walls 60 of each microneedle, as shown in FIG. 8D. The exposedmetal seed layer is then removed using wet etching techniques to exposethe underlying photoresist. Once the metal seed layer has been etched,the wafer is placed in an acetone bath to remove the photoresist frominside the microneedle structures, thereby producing a plurality ofhollow microneedles 28, as shown in FIG. 8E.

In the final processing step, sacrificial membrane 52 is removed by anisotropic etching technique, such as reactive ion etching in a SF₆plasma. Thus, the microneedle ends are released from sacrificialmembrane 52 and are freely suspended, projecting outward from substrate26, as depicted in FIG. 8F and in the embodiment of FIG. 3.

Alternatively, the fabricating method outlined above can be used to forma multi-lumen microneedle by forming bottom walls 54 with zero spacingtherebetween on substrate 26 and carrying out the remaining steps asdescribed above. A resulting multi-lumen microneedle 62 with a pluralityof microchannels 64 therein is shown in FIG. 8G.

In an alternative fabrication method, the microneedle array can bereleased from substrate 26 following surface fabrication on substrate26, such as for the embodiment shown in FIG. 4. This method does notrequire sacrificial membrane formation or KOH etching. Instead, theneedle arrays are released using wet etching of the seed metal fromunderneath the needle structures. If the seed metal is copper, forexample, then this can be done by a selective etch of ammonium hydroxidesaturated with cupric sulfate.

Arrays of two up to hundreds of microneedles can be easily andeconomically fabricated in a package with dimensions on the order ofmillimeters according to the above procedure. A single microneedle suchas shown in FIG. 1B can also be fabricated according to the procedureoutlined above. In addition, instead of having the fluid outlet at thetips of the microneedles, fluid outlet ports can be etched into the sidewalls, top walls, and/or bottom walls of the microneedles if a largeramount of fluid transfer is desired.

Using the above fabrication methods provides the flexibility tointegrate many additional functions directly onto the microneedles.These fabrication methods are low temperature and are compatible withintegrated circuit (IC) technology as a post process. For example,various active components such as sensors, transducers, and electronicinterfaces may be fabricated as integrated components on-chip during theneedle fabrication process. An example of a procedure for fabricating amicroneedle with on-chip devices such as CMOS (complementary metal-oxidesemiconductor) devices is disclosed in U.S. Pat. No. 5,591,139 to Lin etal., the disclosure of which is incorporated by reference herein.Alternatively, the active components may be in the form of prefabricatedchips which are subsequently placed within the needle channels.

All of the needle devices including microneedles and arrays of thepresent invention can be coated on the inside with biocompatiblematerials, such as silicon nitride, gold, plastics, etc., byconventional coating processes known to those skilled in the art.

In addition, a light enhancing reflective coating can be formed on theinner surfaces of the active needle devices defining the lumens therein.The reflective coating can enhance the light output generated during abioluminescent reaction in a bioluminescent-based active needle toprovide for more sensitive biosensing capabilities. Suitable reflectivematerials that can be used to coat the inner surfaces of the needledevices include silver, chromium, titanium, platinum, combinationsthereof, and the like. Increased light intensity has also been observedfor long narrow channels, which can be used to effectively design abioluminescent-based micro-needle. Further details related to lightenhancement of micro reaction chambers and long narrow (micro) channelsare described in Bartholomeusz et al., “BIOLUESCENT BASED BIOSENSOR FORPOINT-OF-CARE DIAGNOSTICS,” First Annual International IEEE Conferenceon Microtechnology, Medicine, and Biology, Lyon, France, Oct. 12-14,2000, the disclosure of which is incorporated by reference herein.

FIGS. 9A and 9B depict alternative methods of assembling two-dimensionalneedle arrays into three-dimensional needle array devices. In the methoddepicted in FIG. 9A, a plurality of two-dimensional microneedle arrays70 are provided which have been released from a substrate as shown forthe array of FIG. 4. The microneedle arrays 70 are positioned in astacked configuration with a plurality of metallic spacers 72therebetween to define the distance between any two microneedle arraysin the stack. The stacked needle array configuration is then subjectedto flash electroplating to join microneedle arrays 70 with metallicspacers 72 in a fixed three-dimensional needle array device 74. Theneedle array device 74 can then be disposed in a machined interfacestructure 76, such as an acrylic interface, allowing connection to adispensing means for injecting a liquid such as a syringe.

In the method depicted in FIG. 9B, a plurality of two-dimensionalmicroneedle arrays 80 are provided on substrates 82 such as shown forthe microneedle array of FIG. 3. The microneedle arrays 80 arepositioned in a stacked configuration, with substrates 82 acting asspacers between arrays 80, to define the distance between any twomicroneedle arrays in the stack. The stacked array configuration isplaced in a mold 84 such as an aluminum mold for plastic injectionmolding. The stacked array configuration, is then subjected to plasticinjection molding. This bonds microneedle arrays 80 together with aplastic molding material 86, thereby forming a fixed three-dimensionalneedle array device 88. The array device 88 can then be disposed in aninterface structure 76 allowing connection to a dispensing device suchas a syringe.

In another alternative method of assembling two-dimensional needlearrays into three-dimensional needle array devices, the two dimensionalarrays are manually assembled under a microscope. The two-dimensionalarrays are stacked with spacers or with substrates on the arrays and arebonded together with a polymeric adhesive such as a UV-curable adhesiveto form a three-dimensional needle array device, which can then bedisposed in an interface structure.

The fabricated three-dimensional needle array devices are typicallydimensioned to have a length of about 5 mm, a width of about 5 mm, and aheight of about 2 mm.

The interface structures for connection to a syringe can be made from avariety of plastic materials such as acrylics, polystyrene,polyethylene, polypropylene, and the like. The interface structuretypically accommodates a three-dimensional needle array device having upto about 25 two dimensional arrays. The interface structures are bondedto the three-dimensional needle array devices with a polymeric adhesivesuch as a UV-curable adhesive. The interface structures are configuredto accept direct syringe connection via a connection means such as aconventional Luer-lock connector. Alternatively, interface structurescan be formed for a single two-dimensional array or a single microneedleso as to accept direct syringe connection via a connector such as aLuer-lock connector.

In alternative embodiments of the invention, any of the above describedarray embodiments can be fabricated as a slanted active needle array,with needles in the array having varying lengths. The slanted needlearray allows for collection of interstitial fluid samples at differentdepths during sample acquisition from a patient. Additionally, theslanted needle array allows for dispensing of medicaments into a patientat multiple depths throughout and beneath the dermal layers of the skin.

Further details regarding suitable microneedle and microneedle arrayssuitable for use in the present invention are described in copendingapplication Serial No. PCT/US99/21509, filed Sep. 17, 1999, entitled“Surface Micromachined Microneedles” and which is incorporated byreference herein.

As discussed above, one or more active components such as a biosensorcan be placed or integrated directly into the needle devices of thepresent invention. A biosensor is basically an analytical device thatconverts the concentration of an analyte in a sample into an electricalsignal by way of a biological sensing element intimately connected witha transducer. A particularly suitable biosensor useful in the needledevices, of the invention is a bioluminescence-based biosensor, whichcan be employed in any of the above described embodiments. Such abiosensor can be formed by depositing biochemical sensing reagents onone or more inner walls of a needle device. For example, variousbisosensing reagents can be drawn into and dried down onto the innerwalls of the hollow microneedle or microneedle arrays describedpreviously. Bioluminescence-based biosensors are particularly suitablefor monitoring metabolic levels, such as the level of creatine orglucose in blood. Generally, bioluminescent detection of the biosensoroutput is performed at the output ports of the microneedle.

Bioluminescence-based chemical analysis has the advantages of beingsubstantially more sensitive than conventional chromogenic (absorbance)measurements and is known to be accurate over a range of five or moreorders of magnitude of concentration ranges. It is known that fireflybioluminescence occurs by enzyme-catalyzed oxidation of luciferinutilizing adenosine triphosphate (ATP). Bacteria bioluminescence isclosely coupled to nicotinaminde adenine dinucleotide hydride (NADH).Bioluminescent-based chemical analysis has the potential of measuring awide range of metabolites from smaller sample volumes.

Since most metabolites in the body are within one or two reactions fromATP or NADH, they can be measured by coupling other enzyme reactions toan ATP or NADH bioluminescent reaction and measuring the light output.During the production or consumption of a metabolite of interest, enzymelinked reactions will cause the production or consumption of ATP (orNADH) through the following bioluminescent platform reactions.

The ATP platform is based on the firefly luciferase reaction:

The NADH platform reaction based on NADH:FMN oxidoreductase andBacterial Luciferase:

Substrates are coupled to these platform reactions through the followinggeneric reaction:

substrate+ADP(or NAD(P)⁺)

product+ATP(or NAD(P)H)

The detected changes in light intensity (measured by various lightdetectors as described below) will be stoichiometrically proportional tothe time changing concentration of the platform molecule (ATP or NADH)and thus proportional to the metabolite of interest. The bioluminescentplatform reactions can be used to measure metabolites such asL-Phenylalanine, D-Glucose, L-Lactate, glucose 6-phosphate, glucose1-phosphate, UDP-glucose, UDP-galactose, lactose, alkaline phosphatase,galactose in solution, human blood (serum & plasma), sweat, and humanmilk.

The light produced in the bioluminescent platform reactions can bedetected by an optical detector such as a photodiode or charge coupleddevice (CCD). Such photosensing devices can be integrated inside amicroneedle to detect the light, or can be located externally and can beused to detect the luminescence through a window in a microneedlesurface. Depending on the instrument used to detect the luminescence,nanomolar, picomolar or even femtomolar analyses are possible. Withincreased sensitivity, smaller amounts of sample fluid are required foraccurate analysis. A 0.05 micro-liter sample size that can be drawn bythe microneedles can painlessly access small amounts of body fluid thatcan be analyzed via bioluminescence.

The transmittance of light through a medium across a certain length (l)is specified through Beer's law, T=10^(−Absorbance), whereAbsorbance=ε_(λ)C_(B)(l-x), and ε_(λ) is the molar absorptioncoefficient in dm³/mole-cm, and C_(B) is the concentration in mole/dm³.Assuming the light travels through a homogeneous medium along lengthmole/dm³. Assuming the light travels through a homogeneous medium alonglength (l), the total transmitted light (T) can be expressed as:

T _(total)(1)=∫_(length)10^(−ε) _(λ) ^(C) _(B) ^((l-x))dx=(−10^(−l))/Ln10

where ε_(λ)C_(B) is assumed to be 1 for simplicity. For a pyramid (whichis the shape of the etched wells of the <100> silicon wafers), where thepeak depth of the pyramid is equal to the length (l) and the base widthof the pyramid is (a), the transmittance is integrated through theviewing area and is equal to T_(total(l))*a²/3. Thus, a linearcorrelation of a volume versus integrated luminosity plot for a givenviewing window should indicate a transmittance proportional to signalintensity (average CCD counts over the viewing area, for example). Aflat bottom well with a reflective surface will have a transmittance ofT_(total(2l)), and will be about twice as large as T_(total(l)) (whenlength<<1^(−cm)). Thus, the slope of a linear fit of volume versusintegrated luminescence intensity for a reflective surface of a givenviewing window should be about twice that of a non-reflective surfacedue to the increased transmittance.

The enzymes suitable for use in the bioluminescent platform reactions ofthe biosensors are inherently robust and stable, retaining theiractivity after lyophilization, deposition, and rehydration. Thereagent/enzyme mixture or “cocktail” used to detect various metabolitesis drawn into the needles after, fabrication, lyophilized (freeze dried)in a stable state, and then packaged for long term storage (up to a yeardepending on the analytes, reagents, and enzymes needed). Thereagent/enzyme mixture includes the ATP and NADH bioluminescent platformenzymes and the enzymes that couple the metabolites to the platforms, aswell as all the necessary reagents needed to complete all the reactionsthat lead to the light output.

The methods used for preparing and storing reagent/enzyme mixturesinside the bioluminescent-based active needle devices are discussed asfollows.

1. Drawing of Reagent/Enzyme Mixtures into the Needle Lumen(s)

The reagent enzyme mixtures or “cocktails” are prepared as a “wet”solution in the concentrations needed to detect the desired analytesfrom the sample volume drawn through the needle lumen. Anti-oxidants andother enzyme stabilizing agents are added to the “cocktail” beforelyophilization. These enzyme-stabilizing agents are described below inthe lyophilization process.

The prepared wet solutions are drawn into the needles by capillaryaction. The filling of the needles is ensured by oxidizing the lumen ofthe needle during the needle fabrication process (or at least using ahydrophilic metal for the lumen of the needle). Each needle lumen has adifferent “cocktail” for measuring different analytes. Reagents andenzyme-stabilizing agents can be added to an agarose solution which isdried during the lyophilization process and allows for the sample fluidto be drawn into the needle faster by capillary action.

2. Lyophilization Process

Lyophilization stabilizes enzymes for long-term storage by reducing bothmechanical and chemical degradation. Appropriate stabilizing excipientsand preservatives minimize the denaturation often observed during theprocesses of lyophilization. Optimized process variables include theinitial concentration of enzyme, buffers that exhibit minimal changes inpH with freezing and drying, freezing rate (should be slow), and variousadditives.

The glass transition temperature of the amorphous phase of thelyophilized enzyme needs to exceed the planned storage temperature ofthe needle devices. Glass transition temperature of the lyophilizedenzyme can be increased by using disaccharides and polymers (used incombination with disaccharides). Disaccharides such as sucrose andtrehalose are especially good at stabilizing the enzyme during freezingand dehydration. The sugar to protein weight ratio should be at leastabout 1 to 1, although stability can be further increased with greatersugar (5 to 1 ratio). Reducing sugars such as glucose, lactose, maltoseor maltodextrins should be avoided because of their tendency to degradeproteins through the Maillard reaction between the carbonyls of thesugar and the free amino groups of the protein. Furthermore, surfactantscan be used to inhibit aggregation at very low concentrations, such asless that about 0.5% per volume.

Antioxidants such as dithiotheitol and glutathione are used duringlyophilization and subsequent storage to prevent oxidation of fireflyluciferase due to sulthydryl groups. Bovine serum albumin can be usedfor surface passivation. Polyethylene oxide (PEO)-based polymers andsurfactants are also effective for surface passivation. Various enzymepreservation and stabilization cocktails are well known and widely usedto maintain enzyme activity under dry storage conditions. In oneembodiment of the invention, bioluminescent reagents are preserved andstabilized with the following components: (1) 0.45 M glycyl glycinebuffer (pH 7.8); (2) 1 mM EDTA; (3) 1 mM dithiothreitol; (4) 10 mMMgSO₄; (5) 1 mg/ml of bovine serum albumin (6) 1 wt % sucrose; and (7) 1wt % Dextran T-40. It has been found that such a mixture is suitable forstabilization of creatine kinase, a particularly delicate enzyme.

The actual bioluminescent molecules (ATP, FMN, bacterial and fireflyluciferase, Oxidoreductase, etc.) are added to the preservativereagents, mixed thoroughly and added to each channel of a pre-chilledbiosensor. In one preferred method, the biosensor and reagents arerapidly frozen to about −70° C. followed by a two-stage lyophilizationprocess. The first stage of lyophilization proceeds for 24 hours atabout −50° C. and less than about 100 mTorr of pressure. The secondstage of lyophilization proceeds for an additional 24 hours at about+30° C. and less than about 100 mTorr of pressure. Air is thenre-admitted to the lyophilization chamber and the biosensors areremoved. Each completed biosensor is then stored in a black plasticcontainer with a gas tight lid that also contains a desiccant and ahumidity indicator membrane. It has been found that this method usedwith firefly luciferase can preserve more than half the enzymaticbioluminescent activity for a minimum of six months.

The interaction between histidine and Ni⁺⁺ can also be used forimmobilization of the enzyme. In biosensor applications, enzymes can beimmobilized on a solid support to prevent diffusion (into the samplesolution) and minimize interference with other channels of the sensor.The performance of the sensor can be adjusted by changing theimmobilized enzyme amount. The recombinant enzymes with BCCP domains canbe immobilized through this interaction with high affinity (Ka=10¹⁵ M⁻¹)There are a variety of solid matrices that can be used forimmobilization.

Co-immobilization of sequentially operating enzymes improves totalreaction efficiency, leading to higher sensitivity. A coupled assay withcoimmobilized luciferase and flavin reductase has the advantage that theFMNH₂ produced from the flavin reductase can be used more effectivelyfor the luciferase reaction, reducing its autooxidation. Traditionalimmobilization methods use chemically conjugated enzymes on solidmaterials, resulting in low immobilization efficiency and low andinconsistent enzyme activity, due to nonspecific immobilization andsurface-induced activity loss.

In one, method of the invention, a biotin-avidin system for proteinimmobilization is utilized, due to its high affinity (K_(a)=10¹⁵/M) andstability. In another method, V. harveyi luciferase and FRase Ibiotinylated is produced in vivo, the proteins are immobilized onavidin-conjugated beads, and the enzyme beads are used to assay NADH. Inusing this method, it has been found that the co-immobilized enzymes hadeight times higher bioluminescence activity than the free enzymes at lowenzyme concentration and high NADH concentration. In addition, theimmobilized enzymes were more stable than the free enzymes. Thisimmobilization method is also useful to control enzyme orientation,which can increase the efficiency of sequentially operating enzymes likethe oxidoreductase-luciferase system.

3. Storage of Active Needle Devices

Storing the bioluminescent-based active needle sensors with thelyophilized “cocktail” in a desiccant will ensure that glass transitiontemperatures of the lyophilized enzyme are greater than storagetemperatures. The active needle devices should be stored in the dark tominimize photooxidation.

Further details regarding bioluminescence-based biosensors as used inthe present invention are set forth in the Examples section below.

Another sensor that can be utilized in the needle device of theinvention is an integrated pressure sensor in, the form of an opticalFabry-Perot cavity, which is depicted in FIG. 10. The pressure sensorcan be integrated into any of the above embodiments by forming a glassplate 120 on one interior surface 121 of a hollow microneedle. A cavity125 is etched in glass plate 120 on the side of the sensor which is tobe in proximity to the fluid flowing in the microchannel of the needle.A thin silicon diaphragm 126 (e.g., about 25 microns thick) is affixedover cavity 125 and is configured to be in direct contact with a fluidwhose pressure is to be measured. The diaphragm 126 deflects in responseto applied pressure. A light beam 129 (e.g., produced by an LED) iscoupled into the sensor through optical fiber 128. The optical fiber 128optically communicates with the interior of the microneedle through awindow 124 in the side of the microneedle. Pressure changes from fluidflowing past diaphragm 126 cause the diaphragm to move. The cavity 125functions as a Fabry-Perot cavity and the diaphragm reflects varyingamounts of light intensity which is directly proportional to the amountof pressure applied to the diaphragm by the fluid. The reflected lightcan be coupled as a signal out 130 through optical fiber 128 to anappropriate analyzing device for measurement. The change in reflectedlight can be measured and with proper calibration, can provide a linearmeasurement of the amount of pressure in the fluid.

The active needle devices of the invention such as the microneedle andmicroneedle arrays have many benefits and advantages. For fluidinjection, these benefits include reduced trauma at penetration sitesdue to their small size, greater freedom of patient movement because ofthe minimal penetration depth of the needles, a practically pain-freedrug delivery due to the smaller cross section of the needle tip anddistribution of fluid force, and precise control of penetration depthfrom needle extension length. In addition, the microneedles have theability to deliver drugs to localized areas, or extract fluids fromprecise locations for analysis, and are advantageous in their ability tobe stacked and packaged into a three-dimensional device for fluidtransfer. The active microneedles have the ability to extract eitherlarge or small amounts of fluids in precisely controlled amounts, andcan be easily and economically fabricated using micromachiningprocedures.

The active microneedles also provide convenient and easy methods forperforming electrical, optical, physical, or biochemical sensing incompact devices. The integrated sensing capabilities in the activemicroneedles provide compact, portable, and disposable devices which areless costly and complex to use for analysis of fluid samples, and reduceor eliminate the need for supporting fluidic and electronic systems.

The microneedles can be used in a wide variety of biomedicalapplications. The microneedles can withstand typical handling and cansubcutaneously deliver medication without the usual discomfortassociated with conventional needles. The microneedles are minimallyinvasive, in that the microneedles only penetrate just beyond the viableepidermis, reaching the capillaries and minimizing the chance ofencountering and damaging the nerves present in the area of penetration.

The following examples are given to illustrate the present invention,and are not intended to limit the scope of the invention.

EXAMPLE 1

A two dimensional microneedle array was fabricated with 25 hollowmicroneedles fluidly interconnected by a needle coupling channel. Thehollow microneedles were made such that their inner cross-sectional areawas 40×20 μm² (width by height) and their outer cross-sectional area was80×60 μm². The needle coupling channel was 100 μm wide, and providedfluid communication between each needle channel. Two sets of 60×100 μmstructural supports were located 250 μm from each needle end. Eachneedle channel was 2 mm long, while the width of the 25 needle array was5.2 mm. The center-to-center spacing between individual needles was 200μm. The needle walls were made of electroformed metal and wereapproximately 20 μm thick.

The microneedle array was fabricated from electroformed low stressnickel sulphamate, gold cyanide, and palladium electroforming solutions.The bath chemistry and electroplating conditions were selected andprecisely controlled to allow formation of low stress depositions on topof a 3-5 μm sacrificial membrane. The surface roughness of theelectroplated metals was found by Atomic Force Microscope (AFM) to beapproximately 15 nm, resulting in a relative roughness of 0.00056.

It is important to note the structural quality of the needle tips. Theinner dimensions were approximately 30×20 μm², outer dimensions wereapproximately 80×60 μm², and the needle tips were formed with 45° anglesfor ease of penetration.

EXAMPLE 2

Individual hollow metallic microneedles were fabricated with multipleoutput ports and had an inner cross-sectional area of 140×21 μm² (widthby height) with outer cross-sectional area dimensions of 200×60 μm². Thetip dimensions of the microneedles were less than 15×15 μm². The lengthof the tapered portion of the needle shaft was 1 mm and the distancefrom the tip to the first output port was about 300 μm. The total lengthof each microneedle was 6 mm, with input port inner dimensions of 140 μmwide and 21 μm high. The wall thickness of each microneedle was about 20μm, and the output ports were formed with dimensions of 30 μm² on thetop and bottom of the microneedles. The output ports were separated by30 μm, and there were 9 ports on the top and 12 ports on the bottom ofeach microneedle.

The microneedles were each packaged into a standard Luer-lock fittingusing a polymeric medical grade UV-curable adhesive. This interfacebetween a microneedle and a syringe included a simple female Luer-to-1/16″ barb adapter. The UV-curable adhesive was found to permanentlyaffix the microneedle to the interface while providing a leak resistantseal.

EXAMPLE 3

A preliminary experiment was conducted to assay a glucose viaglucokinase and firefly luciferase reactions. The assay was ahomogeneous ATP depletion type assay with initial concentrations ofglucokinase (80 nano-molar), ATP (10 micro-molar), luciferin (100micro-molar), and firefly luciferase (0.1 nano-molar) in 0.45 M glycylglycine buffer at pH 7.8. The results are shown in the graph of FIG. 11,which plots the normalized luminometer bioluminescence, detected twominutes after the rapid mixing of all reactants, as a function ofinitial glucose concentration. The results suggest that a single stephomogeneous assay for glucose can be accomplished without initialdilution of the glucose in the sample.

EXAMPLE 4

FIGS. 12 and 13 are graphs showing the characteristics for a creatinebiosensor. The graph of FIG. 12 plots the normalized bioluminescentsignal (in relative light units) as a function of time for severaldifferent concentrations of creatine. The bioluminescent signal had a560 nm wavelength and was measured by a luminometer. The finalconcentrations of creatine shown are within the physiological serumcreatine concentration range.

FIG. 13 shows the results of an assay experiment for creatine using thecreatine kinase and firefly luciferase reactions. The creatine assay wasa homogeneous ATP depletion assay (the creatine kinase reaction competeswith the firefly luciferase reaction for the ATP). The final assayconstituents were buffered in 0.45 molar glycine-glycine buffer at pH7.8 and the final assay concentrations were the following: 1.4 μm ATP,71 μm Mg²⁺, 14 μm luciferin, 0.3 μm luciferase, 60 μm creatine kinase,and various concentrations of creatine (0.45 M glycine-glycine bufferfor control). Relative light units (RLU) measured by the luminometer aredependent on the type of instrument, sensitivity of the instrument, andother factors; therefore, the raw bioluminescence signal measured versustime was normalized to the control peak RLU value. FIG. 13 plots thenormalized bioluminescent signal as a function of creatineconcentration.

EXAMPLE 5

In order to determine the feasibility and the physical limitations ofusing bioluminescence for highly sensitive analyte measurement of smallsample volumes, micro-reaction chambers (μRCs) were fabricated onsilicon wafers using KOH anisotropic wet etching. An ATP fireflyluciferase/luciferin solution was placed in the ARCs and observedthrough a close up lens with a CCD. The integrated CCD signal wasrecorded and compared with well size and depth. The attenuation of theCCD signal was also observed for wafers coated with titanium (500 Å)followed by chromium (1500 Å).

A 5-mL firefly luciferase/luciferin solution consisted of 1.25 mg/mLbovine serum albumin (Sigma−reconstituted into the solution and used forcoating the glass vial to prevent denaturing of the luciferase), 1.25 mMethylene diaminetetra acetic acid (Sigma), 12.5 mM Mg⁺⁺ (Sigma—fromMgSO₄), 1.84-μm firefly luciferase (Promega), and 1.25 mM luciferin(Biosynth) in a 1.25 mM glycyl-glycine buffer. This mixture was able tomaintain 90% activity for about 20 hours when stored in the dark. A 5 mMATP, glycyl-glycine buffer solution was also prepared. A 20 μL sample ofthe firefly luciferase/luciferin solution was pipetted into 5 μL of theATP solution that resulted in a 1.0 mM ATP mixture, saturated withluciferase and luciferin (which means the reaction rate was at itspeak). After the solution mixed, 20 μL of it was pipetted onto an areaabout 20×15 mm wide. A thin glass cover slip was placed on the solution,starting at one end and tilting the cover slip as it was laid down, sothat the excess bioluminescent fluid would disperse. For highconcentrations of ATP (>8 μm), the initial mixing of ATP with theluciferase/luciferin solution causes a peak luminescence within 3seconds. The luminescence then tapers and levels off after 1 minute.Therefore, the light measurements for this experiment were integratedfor 20 seconds, 2 minutes after the ATP and luciferase/luciferinsolution were mixed, with the light intensity essentially constant.

An ST6-A CCD camera, from Santa Barbara Instruments Group was fittedwith an Olympus wide-angle lens and close-up ring. The experimentalsubstrates were focused 55 mm below the lens with the aperture set at2.8. The field of view was about 20×15 mm. The camera was operated at−20.00° C. One minute after the luciferase/luciferin solution was, addedto the ATP, a 10-sec dark field exposure was taken with the CCD. At 2minutes and 5 seconds after the ATP and luciferase/luciferin solutionmixed, the CCD integrated a 20 second exposure, of the bioluminescentreaction while shrouded in a darkroom. The resulting image was saved asa TIF file (Range: 0-400). Scion Image (based on the NIH image software)was used to determine the average and standard deviation of the pixelvalues for each μARC. One pixel of the CCD image was equal to 46.875 μm.Actual results failed to show light emitting from the 75, 50, 25, and 10μm wide wells on all substrates.

FIG. 14 is a graph of the average CCD counts from the 20 secondintegrated CCD reading as a function of different sample volumes. Thedata was plotted in sets for the same viewing area (or μRC squarewidth). The data was also separated according to Ti/Cr reflectivesubstrates and plain, non-reflective substrates. For each set of data(wells with same width and coating), the increasing volume occurs fromthe increased etch depths of 25 μm 100 μm, and 50 μm. The data from thedifferent etch depths are pointed out for the 750 μm wide wells as anexample. The higher intensity values occurred for the deeper wells. Theintensity/volumes slope is greater for the Ti/Cr reflective substratesthan for the plain substrates.

Table 1 below sets forth a statistical comparison of theintensity/volume slope relationships for reflective vs. non-reflectivesurfaces of the same viewing window size. The reflective wells withintensity/volume slopes that were statistically different from theintensity/volume slopes for the non-reflective surfaces (P<0.05 usingthe T-test) were over two times greater. This follows the hypothesisthat transmittance is nearly doubled for chromium coated(reflectivity=0.67@560 nm) versus uncoated silicon(reflectivity=0.33@560 nm) μRCs.

The T-test used was the student-T test to find if there was asignificant difference between sample measurements of two groups. Thenull hypothesis of the T-test (for the described experiment of thisexample) was: there is no difference in the intensity/volume slopes forthe wells with reflective surfaces and the wells with non-reflectivesurfaces. The value P<0.05 means that there is less than a 5%probability (P) that the null hypothesis is true. The T-test, ifperformed properly, is a useful tool to show that one method or productis significantly different (more efficient, better, etc.) than anexisting method or product.

TABLE 1 P value from T-test between Intensity/Volume Slopes of Ratio ofIntensity/Volume μRC Width Non-Reflective μRCs and Slopes for Reflectiveand (μm) Reflective μRCs Non-Reflective Substrates 750 0.00001 3.02 5000.002 2.37 400 0.004 4.94 300 0.005 5.11 250 0.36 2.87 200 0.030 1.07150 0.72 0.88 100 0.55 0.21

The intensity/volume slope also increases as the μRC square widthdecreases. This implies that using long, narrow microneedles as the μRCis ideal. Table 2 statistically compares the intensity/volume slopesbetween reflective wells of different viewing window sizes, showing thatthere is a significant increase in the intensity/volumes slopes, as theμRC width decreases. The intensity/volume slope ratio is the slope forthe smaller window over the slope for the next larger window.

TABLE 2 P value from Ratio of Intensity/Volume μRC Square T-test betweenSlopes for Smaller μRC Widths Being Intensity/Volume Slopes WidthsVersus Wider μRC Compared (μm) μRCs being compared Widths 500/750 0.0041.89 400/500 0.04 1.98 300/400 0.28 1.59 250/300 0.89 1.11 200/250 0.811.28 150/200 0.91 0.73

The increase in intensity/volume is only significant down to wells thatare 400 μm wide. For smaller μRC widths (150-300 μm), the non-reflectivesubstrates show little correlation between average CCD counts and samplevolumes (R²<0.25, where R is the reflectivity). The Ti/Cr reflectivesubstrates still show some correlation (R²>0.25 for ARCs 250 μm wide andwider) between intensity and volume. However, as the μRC square widthdecreases (250 μm wide and smaller), there is little difference insignal intensity values for different etch depths. The error in thesignals for wells 200 μm wide and smaller is large enough to overlapwith the background intensity. This suggests that the wells with windowssmaller than 200 μm wide would not produce signals of a discernableintensity, if they are only 250 μm deep. However, narrower and deeperμRCs such as microneedles, which hold a larger sample volume, wouldproduce a stronger useable signal.

The present invention may be embodied in other specific forms without,departing from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

1. An active needle device, comprising: a hollow non-silicon elongatedshaft defining at a plurality of separated microchannels, said pluralityof microchannels each having a proximal end and a distal end, andwherein said plurality of microchannels are parallel at said proximalend, and wherein said plurality of microchannels merge into a singledistal microchannel within said hollow non-silicon elongated shaft atsaid distal end; at least one input port at said proximal end of each ofsaid plurality of microchannels and at least one output port at a distalend of said distal microchannel, said plurality of microchannels andsaid distal microchannel providing communication between said at leastone input port and said at least one output port; and at least oneactive component in each of said plurality of said parallelmicrochannels.
 2. The active needle device of claim 1, wherein saidsensor comprises a biosensor.
 3. The active needle device of claim 2,wherein said biosensor is a bioluminescence-based biosensor.
 4. Theactive needle device of claim 1, wherein said at least one activecomponent comprises an integrated circuit chip.
 5. The active needledevice of claim 1, wherein said at least one active component comprisesan optical detector.
 6. The active needle device of claim 1, whereinsaid at least one active component comprises a transducer component. 7.The active needle device of claim 1, wherein said at least one activecomponent facilitates taking one or more measurements selected from agroup consisting of biochemical measurements, optical measurements,electrical measurements, physical measurements, and combinationsthereof.
 8. The active needle device of claim 1, wherein at least two ofsaid plurality of microchannels within said hollow non-silicon elongatedshaft are configured to receive different fluids and combine saiddifferent fluids in said distal microchannel.
 9. An active microneedlearray device, comprising: a substrate having a substantially planarfirst surface and an edge adjacent said substantially planar firstsurface; a plurality of hollow non-silicon microneedles positioned onsaid substantially planar first surface of said substrate, each of saidhollow non-silicon microneedles having at least one microchanneltherethrough providing communication between at least one input port ata proximal end of each of said hollow non-silicon microneedles and atleast one output port at an opposite distal end of each of said hollownon-silicon microneedles, wherein said hollow non-silicon microneedlesextend beyond said edge of said substrate and extend in a directionsubstantially parallel to said substantially planar first surface; andat least one active component located in one or more of said hollownon-silicon microneedles.
 10. The microneedle array device of claim 9,wherein said hollow non-silicon microneedles comprise a two dimensionalarray.
 11. The microneedle array device of claim 9, wherein said hollownon-silicon microneedles comprise a three dimensional array.
 12. Themicroneedle array device of claim 9, wherein said hollow non-siliconmicroneedles are composed of a metal material selected from a groupconsisting of nickel, copper, gold, silver, platinum, palladium,titanium, chromium, and alloys or combinations thereof.
 13. Themicroneedle array device of claim 9, further comprising a couplingchannel member that provides fluid communication between said hollownon-silicon microneedles.
 14. The microneedle array device of claim 9,further comprising a pair of structural support members thatmechanically interconnect said hollow non-silicon microneedles and thatprecisely control penetration depth of said hollow non-siliconmicroneedles.
 15. The microneedle array device of claim 9, wherein saidat least one active component comprises a biosensor.
 16. The microneedlearray device of claim 15, wherein said biosensor is abioluminescence-based biosensor.
 17. The microneedle array device ofclaim 9, wherein said microchannels of each of said hollow non-siliconmicroneedles are first microchannels, and wherein each of said hollownon-silicon microneedles have at least one additional microchanneltherein, said additional microchannels being substantially parallel tosaid first microchannels.
 18. An active microneedle array device,comprising: a plurality of hollow non-silicon microneedles each havingat least one microchannel providing communication between at least oneinput port at a proximal end of each of said hollow non-siliconmicroneedles and at least one output port at a distal end of each ofsaid hollow non-silicon microneedles; a first structural support memberinterconnecting said hollow non-silicon microneedles adjacent saidproximal end of said hollow non-silicon microneedles; a secondstructural support member interconnecting said hollow non-siliconmicroneedles adjacent said distal end of said hollow non-siliconmicroneedles; and at least one active component located in one or moreof said microneedles.
 19. The microneedle array device of claim 18,wherein said hollow non-silicon microneedles consists of one of a twodimensional array or a three dimensional array.
 20. The microneedlearray device of claim 18, further comprising a coupling channel memberthat provides fluid communication between said hollow non-siliconmicroneedles.
 21. The microneedle array device of claim 18, wherein saidhollow non-silicon microneedles have a plurality of input ports and/or aplurality of output ports.
 22. The microneedle array device of claim 18,wherein said at least one active component comprises a biosensor. 23.The microneedle array device of claim 22, wherein said biosensor is abioluminescence-based biosensor.
 24. The microneedle array device ofclaim 18, wherein said microchannels of each of said hollow non-siliconmicroneedles are first microchannels, and wherein each of said hollownon-silicon microneedles has at least one additional microchanneltherein, said additional microchannels being substantially parallel tosaid first microchannels.
 25. An active needle array device, comprising:a plurality of hollow non-silicon needles each having at least onechannel that provides communication between at least one input port at aproximal end of each of said plurality of hollow non-silicon needles andat least one output port at an opposite distal end; a coupling channelmember that provides fluid communication between said plurality ofhollow non-silicon needles, said coupling channel member being disposedbetween said at least one input port and said at least one output portof each of said plurality of hollow non-silicon needles; and at leastone active component located in said plurality of hollow non-siliconneedles.
 26. The needle array device of claim 25, wherein said hollownon-silicon needles have a plurality of input ports.
 27. The needlearray device of claim 25, wherein said hollow non-silicon needles have aplurality of output ports.
 28. The needle array device of claim 25,wherein said at least one active component comprises abioluminescence-based biosensor.
 29. The needle array device of claim25, wherein each of said hollow non-silicon needles has at least oneadditional channel therein.
 30. The needle array device of claim 25,wherein each of said hollow non-silicon needles is a macroneedle. 31.The needle array device of claim 25, further comprising a reflectivecoating on an internal surface of at least one of said plurality ofhollow non-silicon needles.